It is known that when 11.alpha.-hydroxy progesterone acid succinate was attached to a protein molecule such as bovine serum albumin (BSA) and administered to an animal, antibodies of high titre very specific to progesterone were produced. This was regarded in the art as a significant breakthrough in steroid immunoassay methodology. The 11.alpha.-hydroxy position in the steroid molecule is remote from the important biological centers of the molecule and generally provides a means for attachment by means of a succinate bridge to protein-like substances, thus to form steriod conjugates which, when administered to animals induce formation of antibodies generally useful in the aforesaid radioimmunoassays. The development of a radioimmunoassay procedure for general steroid determination can be accomplished through antibodies to a steroid hemi-ester protein conjugate wherein the hemi-ester is suitably the hemi-succinate, preferably linked through an ester linkage to the steroid molecule at a position in the molecule that is remote from the biologically functional groups that are the key to the desired discrimination. For example, such a position should be remote from the steroid ring A and D functionality of the sex hormone type of steroid, and from the combination of ring A and D functionality in adrenocortical hormones.
In practice conjugation to the protein is commonly through remote amine functions of protein amino acids, and the number of protein residues is preferably between 10 and 40 per protein molecule. In the past the antibodies produced, commonly after subcutaneous or intradermal administration of the conjugate in Freund's adjuvant to the antibodies-donor, were used in a binding assay using in the conjugate a labeled steroid chosen so as to compete with the steroid to be assayed. The steroids, those that were bound by the antibody and those that were not bound, were separated, commonly by centrifugation after immune precipitation, or by charcoal adsorption, or by filtration when the antibody was chemically bound to discrete particles. The label associated with one or both fractions was counted, and the amount of steroid being assayed was calculated using standard calibrated curves.
This prior method ordinarily required a labeled steroid in the conjugate that corresponded to each steroid that was being assayed.
Because the preparation of individual labeled steroids is difficult and costly, often requiring multi-step syntheses using labeled starting materials, a need has rapidly developed for a simpler, less costly method for associating radioactivity with the steroid to be assayed.
It is known that the tyrosine methyl ester amides of certain steroid hemisuccinates could be radioiodinated and could serve then as the label-bearing steroid in a radioimmunoassay procedure. The steroid hemisuccinates were at C.sub.17 in testosterone, at C.sub.21 in aldosterone, corticosterone, cortisone, and deosycorticosterone, and at C.sub.3 in pregnenolone and estradiol (Niswender and Midgley in Piron and Caldwell "Immunologic Methods in Steroid Determination," Appleton-Century-Crofts, 1970, p. 149). Steroid hemisuccinates were conjugated to tyrosine methylester using the method of Oliver et al., J. Clin. Invest. 47, 1035, 1042 (1968) and were radioiodinated by the method of Niswender et al., Endocrinol. 84, 1166 (1969).